KR102560734B1 - Method for preparing Centella asiatica Extracts by three-step fermentation and Cosmetic composition comprising thereof - Google Patents

Abstract

The present invention relates to a method for preparing a fermented centella asiatica extract having an increased content of immunoactive substances through a three-step fermentation process and a cosmetic composition comprising the same. According to the present invention, the centella asiatica fermented extract obtained through the three-step fermentation process has a significantly increased content of immunoactive substances such as madecassoside, asiaticoside, madecassic acid, and asiatic acid compared to organic solvent extraction, and the three-step fermentation The cosmetic composition containing the fermented centella asiatica extract through the process is useful because it has a bioimmunity enhancing effect as well as skin immunity.

Description

Method for preparing centella asiatica extract through three-step fermentation and cosmetic composition containing centella asiatica extract

The present invention relates to a method for preparing a fermented centella asiatica extract having an increased content of immunoactive substances through a three-step fermentation process and a cosmetic composition comprising the same.

Skin aging can be caused by the activation of reactive oxygen species according to ultraviolet rays, stress, disease conditions, environmental factors, wounds, and aging. When this condition intensifies, the antioxidant defense network present in the body is destroyed, cells and tissues are damaged, and skin aging is accelerated. Specifically, lipids, proteins, polysaccharides, and nucleic acids, which are major constituents of the skin, are oxidized to destroy skin cells and tissues, resulting in skin aging. Oxidation of protein cuts skin connective tissue such as collagen, hyaluronic acid, elastin, proteoglycan, fibronectin, etc., causing a severe excessive inflammatory reaction and affecting skin elasticity. If it gets worse, it leads to mutations, induction of cancer, and deterioration of immune function due to DNA mutation.

Centella asiatica is a perennial plant commonly found in the mountains and fields of the southern islands of the Korean Peninsula. The main stem extends to the side, and there are 2 degenerated scale-like leaves near the root node. Leaves grow densely at nodes, elongated, 2-5cm in diameter, glossy surface, with dull sawtooth on the edge, petiole 4-20cm long. Flowers are reddish-purple, 3 to 4 flowers hang in the axils in an umbel, and are shaped like heads. The style is two-pronged, and the fruit is the oblong shape of the branch. In India, when a wounded tiger was treated by rolling around in a place where there were many centella asiatica plants, it was called tiger grass, and it has been used as a medicine for a long time.

Madecassoside, asiaticoside, madecassic acid, and asiatic acid are known as the main active ingredients contained in Centella Asiatica. Among them, madecassoside and asiaticoside are It is a pentacyclic triterpene glycoside belonging to the α-amyrin-ursolic acid group and is used to treat skin wounds or chronic ulcers. Asiaticoside has antibacterial and antifungal properties and has been reported to have therapeutic effects on skin diseases, wounds, gastric ulcers, tuberculosis, venous diseases, dementia, etc. (Ju, JC, Shin, JH, Lee, SJ, Cho , HS and Sung, NJ 2006. Antioxidative activity of hot water extracts from medicinal plants. J. Kor. Soc. Food Sci. Nutr. 35, 7-14). In addition, studies have been reported that derivatives derived from asiaticoside of Centella asiatica protect cells from β-amyloid-induced cell death, and madecassoside and asiaticoside are more effective in healing wounds than madecassic acid and asiatic acid. (Fang Wu, Difei Bian, Yufeng Xia, Zhunan Gong, Qian Tan, Jiaojiao Chen, and Yue Dai. 2012. Identification of Major Active Ingredients Responsible for Burn Wound Healing of Centella asiatica Herbs. Volume 2012 |Article ID 848093 | https://doi.org/10.1155/2012/848093 ).

On the other hand, domestic centella asiatica contains 5.3 times more asiaticoside than Indonesian centella asiatica, which is very good for industrial use of domestic useful resources. As a result of analyzing the components using centella asiatica cultivated in the Chungju region of Korea, it was reported that 14.9mg/g of madecassoside and 32.2mg/g of asiaticoside were detected in the EtOH 70% extract (Hin, HYH Kim, Jeong, EJ Kim, JE Lee , KH Bae, YJ and Yu, KW Yu 2020. Bioactive Compounds, Anti-Oxidant Activities and Anti-Inflammatory Activities of Solvent Extracts from Centella asiatica Cultured in Chungju Korean J. Food Nutr. Vol. 33. No. 6, 692~701 ).

Madecassic acid is a pentacyclic triterpenoid physiologically active substance that has anti-inflammatory and anti-diabetic effects. It alters lipid metabolism, blocks NF-кB activation in macrophages, and is known to be effective against cancer cell lines and several immune genes (Gunapathy Devi, Chellam Balasundaram). , Ramasamy Harikrishnan . 2020. Effect of madecassic acid on innate-adaptive immune response and cytokine gene expression in Labeo rohita against Argulus siamensis. The upper layer of Centella asiatica was collected and an experiment was conducted on the brain of a rat. As a result, it has good inhibitory power of acetylcholine esterase in the autonomic ganglion and the organ that controls the parasympathetic nerve (Pham et al., 2019). Side has been reported to have a good effect on hyperpigmentation or skin whitening (Kwon et al., 2014).

The present inventors have tried to study a method capable of increasing the content of immunoactive substances contained in centella asiatica. As a result, the centella asiatica is fermented in two steps with a specific strain, and then the centella asiatica is mixed with heterogeneous strains and the centella asiatica is fermented in 3 steps. A method for producing centella asiatica extract was completed, and at this time, it was confirmed that the content of the immunoactive substance of centella asiatica greatly increased, and the present invention was completed.

The technical problem to be achieved by the present invention is a three-step fermentation process in which centella asiatica is first fermented with a strain of the genus Bacillus, followed by secondary fermentation with a strain of the genus Lactobacillus, and then third fermentation is performed with two strains of the genus Lactobacillus. It is to provide a method for preparing a centella asiatica extract with an increased content of immunoactive substances through and a cosmetic composition containing the centella asiatica extract.

The technical problem to be achieved by the present invention is not limited to the above-mentioned technical problem, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above technical problem, one embodiment of the present invention is a) centella asiatica (Centella asiatica) is washed and then dried and then pulverized; b) sterilizing the pulverized centella asiatica and then allowing it to cool; c) a primary fermentation step of inoculating the sterilized centella asiatica with bacteria of the genus Bacillus for solid fermentation; d) a secondary fermentation step of inoculating an intermediate product of the genus Lactobacillus for solid fermentation in which the primary fermentation is completed; e) a tertiary fermentation step of inoculating the intermediate product after the secondary fermentation with two strains of the genus Lactobacillus for solid fermentation; and f) extracting by adding an extraction solvent to the fermentation product of Centella asiatica after the tertiary fermentation is completed.

In the present invention, the primary fermentation may be inoculated with 8 to 12 parts by weight of Bacillus subtilis based on 100 parts by weight of Centella Asiatica and fermented at 28 to 32 ° C. for 6 to 8 days.

In the present invention, the secondary fermentation may be inoculated with 8 to 12 parts by weight of Lactobacillus casei based on 100 parts by weight of the primary fermentation product and fermented at 28 to 32 ° C. for 6 to 8 days. .

In the present invention, the tertiary fermentation is carried out by inoculating 8 to 12 parts by weight of Lactobacillus casei and Lactobacillus mesenteroides with respect to 100 parts by weight of the secondary fermentation product to inoculate 28 to 12 parts by weight of Lactobacillus mesenteroides. It may be fermented for 6 to 8 days at 32 ° C.

In the present invention, the immunoactive substance may be selected from the group consisting of madecassoside, asiaticoside, madecassic acid and asiatic acid.

In the present invention, the sterilization of step b) may be sterilization for 10 to 50 minutes at 110 to 130 ° C. and 1 to 2 atm by mixing centella asiatica and distilled water in a weight ratio of 1: 1.

In another embodiment, the present invention provides a cosmetic composition containing the centella asiatica extract prepared by the above method.

In the present invention, the cosmetic composition is skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nutrient lotion, cream, eye cream, massaging cream, nutrient cream, moisture cream, hand cream, foundation, essence , nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, and may be any one formulation selected from the group consisting of, and the Centella Asiatica extract is 1 to 5% by weight based on the total weight of the cosmetic composition. may be included.

According to the present invention, the centella asiatica fermented extract obtained through the three-step fermentation process has a significantly increased content of immunoactive substances such as madecassoside, asiaticoside, madecassic acid, and asiatic acid compared to organic solvent extraction, and the three-step fermentation The cosmetic composition containing the fermented centella asiatica extract through the process is useful because it has a bioimmunity enhancing effect as well as skin immunity.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the description of the present invention or the configuration of the invention described in the claims.

Figure 1 is a photograph of the state during inoculation of primary fermentation ( Bacillus subtilis ), secondary fermentation ( Lactobacillus casei ), and tertiary fermentation ( Lactobacillus casei + Lactobacillus mesenteroides ) strains.
Figure 2 confirms the results of the first day of liquid fermentation in three stages of centella asiatica.
Figure 3 confirms the results of the 4th and 7th days of solid fermentation in three stages of centella asiatica.
4 is a calibration curve of the active ingredient of Centella asiatica as a result of HPLC analysis.
5 shows the chromatogram results of the active ingredient of Centella asiatica as a result of HPLC analysis.

Hereinafter, the present invention will be described in more detail. However, the present invention can be implemented in many different forms and the present invention is not limited by the embodiments described herein.

Terms used in this specification are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In the specification of the present invention, 'include' a component means that other components may be further included without excluding other components unless otherwise stated.

Fermentation means that microorganisms such as yeast or bacteria decompose organic compounds by the comprehensive action of external environments such as temperature, humidity, time, space, etc., and the components are decomposed from high molecules to low molecules. When the strain is first fermented, a fermented product according to the fermentation conditions is produced as an intermediate product, and when it is secondarily fermented with another specific strain, a fermented product is produced according to the fermentation conditions. When the intermediate product of which the secondary fermentation is completed is fermented with two other specific strains, the final fermentation product through three stages is produced according to the fermentation conditions. As such, the fermentation results are different depending on various fermentation conditions such as strain, temperature, humidity, and time.

According to the present invention, the solid fermentation of centella asiatica can be carried out for 6 to 8 days at 28 to 32 ° C. for all of the first, second and third fermentation. At this time, during the first fermentation, the strain of the genus Bacillus is 8 to 12% by weight of centella asiatica, during the second fermentation, the strain of the genus Lactobacillus is 8 to 12% by weight of the product of the first fermentation, and during the third fermentation, the strain of the genus Lactobacillus of the different species is It can be inoculated with 8 to 12% by weight of the secondary fermentation product. The tertiary fermentation is heterogeneous fermentation, in which two strains of the genus Lactobacillus can be inoculated at the same time, and the strains of the genus Lactobacillus may be homogeneous, such as Lactobacillus casei and Lactobacillus mecenteroides. When only one of Lactobacillus casei or Lactobacillus mesenteroides is inoculated in the tertiary fermentation process, the immunoactive material enhancement effect as in the present invention is not shown. Preferably, all of the first, second and third fermentation can be carried out at 30 ° C. for 7 days, the first fermentation strain is Bacillus subtilis, 10% by weight of Centella asiatica, and the second fermentation strain is Lactobacillus caseiro 1 It can be inoculated with 10% by weight of the primary fermentation intermediate, and the tertiary fermentation can be inoculated with Lactobacillus casei and Lactobacillus mesenteroides heterogeneous strains, each with 10% by weight of the secondary fermentation intermediate.

According to the three-step centella asiatica fermentation extraction process according to the present invention, the first fermentation by Bacillus subtilis strain, the second fermentation by Lactobacillus casei strain, and the third fermentation by heterogeneous strains of Lactobacillus casei and Lactobacillus mesenteroides As a result of the sequential fermentation, the content of the immunoactive substance of centella asiatica is greatly increased in the three-stage fermentation extract.

In the present invention, it is preferable to pulverize centella asiatica to 2 to 3 mm, which was confirmed to be the most suitable size for the growth of bacteria.

In the present invention, solid fermentation means fermentation production by microorganisms as a raw material in a solid state with low moisture, and has advantages such as less bacterial contamination and easy waste disposal compared to liquid fermentation.

In the present invention, the term immunoactive substance refers to a substance that enhances the body's immune function, and the immunoactive substance contained in Centella Asiatica is madecassoside, asiaticoside, madecassic acid and It may be asiatic acid. Chemical structures are shown in Formulas 1 to 4 below.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

On the other hand, the cosmetic composition according to one embodiment of the present invention is not limited to being prepared in any formulation commonly prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, It may be formulated as lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be applied to the manufacture of functional cosmetics that are basic cosmetics such as lotion, nourishing cream, moisture cream, essence, eye cream, lotion, pack, and essence. On the other hand, the ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the fermented extract as an active ingredient, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. Conventional adjuvants and carriers may be included. On the other hand, according to one embodiment of the present invention, the fermented Centella Asiatica extract may be contained in an amount of 0.1 to 5% by weight based on the total weight of the cosmetic composition, and has an effect of preventing and improving skin wrinkles within the weight% range, and the Below the weight% range, there may be a problem in that the effect of preventing and improving skin wrinkles is insignificant.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

preparation of centella

Dried centella asiatica was purchased from Hyoun Tech. After purchasing the material, it was pulverized using a grinder (Hanil Hoogmix SMFP-3000) to a size of about 2 to 3 mm to allow the bacteria to grow well, and then the fermented strain was inoculated using this to conduct an experiment.

Preparation of strains

A total of three strains were used in the experiment. The primary fermentation was Bacillus subtilis , the secondary fermentation was Lactobacillus casei , and the 3rd fermentation was Lactobacillus casei for heterogeneous fermentation ( Lactobacillus casei ) and Lactobacillus mesenteroides were used. All strains were distributed in ampoules from the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology, stored in a cryogenic freezer at -80 ° C, inoculated into MRS agar plates, and incubated at 30 ° C before use. During culture, an incubator (JISICO, J-NSIL-R, Korea) was used. All fermentation conditions were carried out using a liquid medium. Solid fermentation was inoculated with 10% (v/w) of the mass fraction, and liquid fermentation was inoculated with 8% (v/w) of the total medium including the culture medium. At the time of inoculation, strains were used that were very active (Fig. 1).

Fermentation process after inoculation of Centella asiatica strain

In liquid fermentation, the primary fermentation strain Bacillus subtilis was inoculated, fermented for 1 day, filtered three times with a bacteria removal filter (HYUNDAI Micro Co., MEMBRANE FILTER CA045047A, 0.45 μm), and then secondary fermentation was performed. The secondary fermentation was performed using the strain Lactobacillus casei, filtered three times with a sterilization filter after fermentation, and then the tertiary fermentation was carried out in an incubator. For the tertiary fermentation, heterogeneous fermentation was performed by mixing Lactobacillus casei and Lactobacillus mesenteroides based on the results of the 1st and 2nd fermentation, and the activity of the strain was observed 1 day after inoculation (FIG. 2 ).

In solid state fermentation, the activity of the strains was observed 4 days and 7 days after inoculation with Bacillus subtilis in the first fermentation, and 4 days and 7 days after inoculation with Lactobacillus casei in the secondary fermentation. Comparative observations were made. After that, the tertiary fermentation activity was observed 4 days and 7 days after inoculation of Lactobacillus casei and Lactobacillus mecenteroides heterogeneous strains (FIG. 3).

Preparation of Centella asiatica fermented extract

For solid fermentation, centella asiatica and distilled water were mixed at a ratio of 1:1 (w/v), sterilized at 121°C, 1.5 atm, and 30 minutes, and then allowed to cool. After activating the bacteria on an agar plate, inoculate 10% (v/w) primary fermented cell culture (Bacillus subtilis) into the sterilized Centella Asiatica by culturing it in a liquid medium, mix well, ferment at 30 ° C for 7 days, , The primary fermentation product was inoculated with 10% (v/w) of secondary fermentation bacteria (Lactobacillus casei) and fermented at 30° C. for 7 days. Thereafter, the secondary fermentation product was inoculated with 10% (v/w) of two tertiary fermentation bacteria (Lactobacillus casei and Lactobacillus mesenteroides), and fermented at 30° C. for 7 days.

Liquid fermentation mixes 1,000g of centella asiatica with 11,500mL of the culture medium of each bacteria at each stage, sterilizes it at 121℃, 1.5 atm, and 30 minutes, then inoculates 1,000mL of bacterial culture, mixes well, and ferments at 30℃ for 1 day At this time, the centella asiatica was inoculated with a primary fermentation fungus (Bacillus subtilis) and fermented, then the primary fermentation product was inoculated with a secondary fermentation fungus (Lactobacillus casei) and fermented, and finally secondary fermentation The product was inoculated with two tertiary fermentation bacteria (Lactobacillus casei and Lactobacillus mesenteroides) and fermented (Table 1).

Stage 3 Fermentation Conditions Furtherance culture conditions temperature number of days control Centella Asiatica 1,000g room temp. 2 days solid
fermentation Centella Asiatica 1,000g + B. subtilis 100ml 30℃ 7 days 1st fermentation product 1,000g + L. casei 100ml 30℃ 7 days 2nd fermentation product 1,000g + L. casei and L. mesenteroides culture medium 100ml each 30℃ 7 days Liquid
fermentation Centella Asiatica 1,000g + TSB 11,500mL + B. subtilis 1,000ml 30℃ 1 day 1st fermentation product 1,000g + MRS 11,500mL + L. casei 1,000ml 30℃ 1 day 2nd fermentation product 1,000g + MRS 11,500mL + L. casei and L. mesenteroides 1,000ml each 30℃ 1 day

After that, the solid fermented Centella asiatica was used with 5 times 70% ethanol (EtOH), and the liquid fermentation of Centella asiatica was made into a 50% ethanol extract at a ratio of 1: EtOH 100% 1 to room temperature. was extracted for 48 hours. As a control, dried centella asiatica was immersed in 70% ethanol (EtOH) and extracted at room temperature for 48 hours. The extracted extract was stored in a refrigerator after removing solids using filter paper, secondary and tertiary filtering. The fermented extract was filtered three times using a sterilization filter, observed under a microscope, and after confirming that the bacteria were completely removed, it was concentrated using a rotary pressure reducer (Rotavapor R-215, Buchi, Swiss). Concentration conditions were vacuum dried under conditions of water bath 45 ℃, 150 rpm. After vacuum drying, the powder was subdivided into 50 ml tubes, stored in a cryogenic freezer at -80 ° C, and then used for experiments.

High-Performance Liauid Chromatography (HPLC) analysis method

The equipment used for the analysis was an HPLC system (Nexera lite, Shimadzu, Japan) consisting of a Solvent Delivery pump, Degasseing Unit, Autosampler, and UV-VIS Detector. A Mightysil RP-18 GP column (4.6 × 250 mm, 5 μm, Kanto Chemical, Japan) was used as the column. Gradient elution was performed under each fermentation condition, and the detection wavelength was detected at 206 nm. The test solution was used after filtering through 0.45 μm. Four standard reagents, madecassoside, asiaticoside, madecassic acid, and asiatic acid, were purchased from Sigma Aldrich (USA) and used.

HPLC analysis conditions equipment condition Column Mightysil RP-18GP 4.6×250mm (5μm) Mobile phase A solvent: 0.5% acetic acid in water
B solvent: acetonitrile
22%B (0-15min),
22-24% B (15-20min)
24%B (20-28min)
24-40% B (28-32min)
40%B (32-45min)
40-46% B (45-48min)
46-70%B (48-50min)
70% B (50-60min)
70-22% B (60-60.1min)
22%B (60.1-61min) Detector 205 nm Flow rate 1.0 mL/min Column temperature 30℃ Injection volume 10 μL

Example 1. Confirmation of active ingredients of 3-step fermented centella asiatica extract

As a result of HPLC analysis, the calibration curve of madecassoside, the indicator component of centella asiatica, was 2745.5x + 3494.1 (r² = 0.999), the calibration curve of asiaticoside was 2839.1x + 20037 (r² = 0.986), and the calibration curve of madecassic acid was 6136.2x + 34280 (r² = 0.997), and the calibration curve of asiatic acid was 4852.8x + 107396 (r² = 0.980), showing good linearity (Fig. 4), and the retention time (R.T) in the HPLC chromatogram was approximately It was confirmed in the 23.60, 33.40, 44.42, and 52.22 squads (Fig. 5).

Example 2. Comparison of Active Ingredient Content of Control and 3-Step Fermented Centella Asiatica Extract

As a control, the results of analyzing the contents of the four active ingredients contained in the centella asiatica extract extracted at room temperature for 48 hours using 70% EtOH at room temperature were analyzed as shown in Table 3, madecassoside 3.99ppm, asiaticoside 9.25ppm , it was confirmed that madecassic acid 29.42ppm, and asiatic acid 107.64ppm were contained.

Active ingredient of centella ethanol extract (EtOH 70%) (unit : ppm) active ingredient Madecassoside Asiaticoside Madecassic acid asiatic acid Total content 3.99 9.25 29.42 107.64 150.3

Table 4 shows the results of the change in the content of active ingredients after performing the first, second and third fermentation (solid fermentation, liquid fermentation) using three strains of centella asiatica. In solid fermentation, the content of madecassoside was 2.60 ppm in the first fermentation and 2.05 ppm in the second fermentation, which was lower than 3.99 ppm in the control ethanol extraction. On the other hand, in the tertiary fermentation, it was 6.74 ppm, showing about three times higher content in the tertiary fermentation than in the 1st and 2nd fermentation. The content of asiaticoside was detected as 31.00 ppm in the first fermentation and 28.64 ppm in the second fermentation, but it was confirmed that the content of 24.04 ppm was slightly lower than that in the first and second fermentations in the third fermentation and heterogeneous fermentation. On the other hand, madecassic acid showed almost similar contents of 24.52ppm and 24.31ppm in the first and second fermentation, and contained 61.64ppm in the third fermentation, which is about 2.5 times higher than the first and second fermentation. seemed It was confirmed that asiatic acid contained 116.68 ppm during the first fermentation, 152.39 ppm after the second fermentation, and 151.70 ppm after the third fermentation.

Next, in liquid fermentation, madecassoside is 3.85 ppm in primary fermentation, secondary fermentation and In the third fermentation, 5.45ppm and 5.49ppm were confirmed. Asiaticoside was 5.38ppm, 6.88ppm, and 6.25ppm in the first, second, and third fermentations, indicating that it had the lowest content during the first fermentation. In addition, madecassic acid was found to be insignificant at 16.42 to 16.87 ppm in all of the first, second and third fermentations. Asiatic acid was found to be 90.80 to 92.75 ppm without significance in all of the first, second and third fermentations.

Changes in active ingredient content of centella asiatica after stage 3 fermentation (unit : ppm)
Madecassoside Asiaticoside Madecassic acid asiatic acid Total solid
fermentation 1st fermentation 2.60 31.00 24.52 116.68 174.8 2nd fermentation 2.05 28.64 24.31 152.39 207.39 3rd fermentation 6.74 24.04 61.64 151.70 244.12 Liquid
fermentation 1st fermentation 3.85 5.38 16.82 92.75 118.80 2nd fermentation 5.49 6.88 16.42 90.80 119.59 3rd fermentation 5.45 6.25 16.87 90.99 119.56 Primary fermentation ( B. subtilis ), secondary fermentation ( L. casei ), tertiary fermentation ( L. casei + L. mesenteroides )

As a result of comparing the content of the control group in which each active ingredient of centella asiatica was extracted with ethanol and the content of the 3rd fermentation, madecassoside was 3.99ppm in the control group, but increased by about 1.69 times to 6.74ppm when treated with solid fermentation up to the 3rd fermentation. , Asiaticoside was 9.25ppm in the control group, but increased to 24.04ppm after the 3rd stage solid fermentation. In addition, in the case of madecassic acid, the control group showed 29.42ppm, and after the 3rd stage solid fermentation, the content was about 2.1 times more than 61.64ppm. Asiatic acid was found to contain 107.64 ppm in the control group, and after the 3rd stage solid fermentation, the content was 151.70 ppm, which was about 1.4 times higher.

As a result of comparing the total active ingredient content of the four types of centella asiatica extracts extracted with an organic solvent and the centella asiatica extract treated with 3-step fermentation, the 3-step liquid fermentation was the lowest at about 120ppm, and the next ethanol-extracted control was 150.30ppm. In the case of solid fermentation, it was found to be 244.12 ppm.

Example 4. Preparation of cosmetics

The composition ratios of finished cosmetic products including madecassoside, asiaticoside, madecassic acid, and asiatic acid, which are four active ingredients included in centella asiatica fermentation extraction, are shown in Tables 5 to 8 below. The composition ratio of the eye cream, essence, and cream formulations was the best when the centella asiatica fermented extract was included at 2%, and no coagulation with other substances was observed. When the formulation composition ratio of the toner was 3% of the fermented centella asiatica extract, the effect of permeating into the skin was stable and there was no clogging with other substances.

Eye cream cosmetic formulation composition ratio containing 3-step fermented centella asiatica extract eye cream ingredient name function content(%) Purified water skin conditioning agent To 100 Butylene Glycol skin conditioning agent 7.00 glycerin skin conditioning agent 6.00 Cetearyl Alcohol emulsion stabilizer 2.80 Hydrogenated polydecene skin conditioning agent 2.50 Centella asiatica fermented extract irritation relief 2.00 1,2-Hexanediol solvent 2.00 shea butter skin conditioning agent 2.00 dimethicone skin conditioning agent 1.00 Arachidyl Alcohol emulsifier 0.54 Glyceryl Stearate emulsifier 0.52 Polysorbate 60 emulsifier 0.50 PEG-100 Stearate Surfactant-cleaning agent 0.48 Glyceryl Stearate SE emulsifier 0.40 Cetearyl Olivate Surfactants 0.30 behenyl alcohol emulsifier 0.28 Sorbitan Olivate Surfactants 0.20 caprylyl glycol skin conditioning agent 0.20 arachidyl glucoside emulsifier 0.18 Stearic Acid emulsifier 0.10 Acrylates/C10-30 Alkyl Acrylate Crosspolymer thickener 0.10 Hydroxyethyl Acrylate/Sodium Acryloyl Dimethyl Taurate Copolymer thickener 0.10 tromethamine pH modifier 0.08 Ethylhexylglycerin skin conditioning agent 0.05 xanthan gum thickener 0.05 Disodium EDT chelating agent 0.02

Composition ratio of toner cosmetic formulations containing 3-step fermented centella asiatica extract toner ingredient name function content(%) Purified water skin conditioning agent To 100 Butylene Glycol skin conditioning agent 5.12 glycerin skin conditioning agent 3.50 Centella asiatica fermented extract irritation relief 3.00 glycereth-26 moisturizer 2.50 propanediol solvent 0.80 PEG-60 Hydrogenated Castor Oil Surfactants 0.40 caprylyl glycol skin conditioning agent 0.20 clophenesin disinfectant 0.20 arginine skin conditioning agent 0.12 Ethylhexylglycerin skin conditioning agent 0.05 Disodium EDT chelating agent 0.05 carbomer thickener 0.05

Composition ratio of essence cosmetic formulations containing centella asiatica 3-step fermented extract essence ingredient name function content(%) Purified water skin conditioning agent To 100 mineral oil skin conditioning agent 6.00 Butylene Glycol skin conditioning agent 3.11 Centella asiatica fermented extract irritation relief 2.00 Caprylic/Capric Triglyceride skin conditioning agent 2.00 glycerin skin conditioning agent 2.00 Stearic Acid emulsifier 0.90 Glyceryl Stearate emulsifier 0.80 Cetearyl Alcohol emulsion stabilizer 0.80 Polysorbate 60 emulsifier 0.50 Glyceryl Stearate emulsifier 0.40 Sorbitan Stearate emulsifier 0.40 PEG-100 Stearate Surfactant-cleaning agent 0.40 phenoxyethanol preservative 0.30 dimethicone skin conditioning agent 0.20 arginine skin conditioning agent 0.20 caprylyl glycol skin conditioning agent 0.20 clophenesin disinfectant 0.20 carbomer thickener 0.16 Ethylhexylglycerin skin conditioning agent 0.05 Disodium EDT chelating agent 0.05

Composition ratio of cream cosmetic formulations containing active ingredients of centella asiatica 3-step fermented extract cream ingredient name function content(%) Purified water skin conditioning agent To 100 Butylene Glycol skin conditioning agent 7.00 Centella asiatica fermented extract stimulant 2.00 glycerin skin conditioning agent 1.50 Arachidyl Alcohol emulsifier 1.08 Cetearyl Alcohol emulsion stabilizer 1.00 isohexadecane skin conditioning agent 1.00 Caprylic/Capric Triglyceride skin conditioning agent 0.80 Cetearyl Olivate Surfactants 0.60 behenyl alcohol emulsifier 0.56 dimethicone skin conditioning agent 0.50 Stearic Acid emulsifier 0.50 Sorbitan Olivate Surfactants 0.40 arachidyl glucoside emulsifier 0.36 arginine skin conditioning agent 0.33 carbomer thickener 0.30 caprylyl glycol skin conditioning agent 0.20 clophenesin disinfectant 0.20 Dipropylene Glycol solvent 0.15 Ethylhexylglycerin skin conditioning agent 0.05 C12-15 Alkylbenzoate skin conditioning agent 0.05 Disodium EDT chelating agent 0.03

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (9)

a) washing Centella asiatica, drying it, and grinding it;
b) sterilizing the pulverized centella asiatica and then allowing it to cool;
c) a primary fermentation step of solid fermentation by inoculating Bacillus subtilis in the sterilized centella asiatica;
d) a secondary fermentation step of inoculating the intermediate product upon completion of the primary fermentation with Lactobacillus casei for solid fermentation;
e) Lactobacillus casei and Lactobacillus mesenteroides in the intermediate product after the secondary fermentation is inoculated with two types of bacteria to solidify the tertiary fermentation step; and
f) A method for producing an extract of Centella asiatica with an increased content of immunoactive substances comprising the step of extracting by adding an extraction solvent to the fermented centella asiatica after the tertiary fermentation is completed.
According to claim 1,
The primary fermentation is to inoculate 8 to 12 parts by weight of Bacillus subtilis with respect to 100 parts by weight of centella asiatica and ferment at 28 to 32 ° C for 6 to 8 days, centella asiatica with increased immunoactive substance content Method for preparing the extract.
According to claim 1,
The secondary fermentation is inoculated with 8 to 12 parts by weight of Lactobacillus casei based on 100 parts by weight of the primary fermentation product and fermented at 28 to 32 ° C. for 6 to 8 days, the immunoactive substance content A method for preparing an increased Centella asiatica extract.
According to claim 1,
The tertiary fermentation is carried out by inoculating 8 to 12 parts by weight of Lactobacillus casei and Lactobacillus mesenteroides with respect to 100 parts by weight of the secondary fermentation product, and inoculating two types of bacteria at 28 to 32 ° C. A method for producing a centella asiatica extract with increased immunoactive substance content, which is fermented for 8 days.
According to claim 1,
Characterized in that the immunoactive substance is selected from the group consisting of madecassoside, asiaticoside, madecassic acid and asiatic acid, the content of the immunoactive substance increases. Method for preparing centella asiatica extract.
According to claim 1,
The sterilization in step b) is to prepare a centella asiatica extract with increased immunoactive substance content, which is prepared by mixing centella asiatica and distilled water at a weight ratio of 1:1 and sterilizing at 110 to 130 ° C. and 1 to 2 atm for 10 to 50 minutes. How to.
A cosmetic composition comprising the centella asiatica extract prepared by the method of claim 1.
According to claim 7,
The cosmetic composition is skin lotion, skin softener, skin toner, lotion, milk lotion, moisture lotion, nutrient lotion, cream, eye cream, massage cream, nutrient cream, moisture cream, hand cream, foundation, essence, nutrient essence, pack , Soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser will be any one formulation selected from the group consisting of, a cosmetic composition.
According to claim 7,
The centella asiatica extract is contained in 1 to 5% by weight relative to the total weight of the cosmetic composition, the cosmetic composition.